Trophic roles: Development and Wound Healing

Tooth Development -Several studies have suggested that nerves in the vicinity o f the developing teeth could influence the surrounding tissues and initiate tooth development (Chiego 1995). This concept seems to be substantiated in a study where development and mineralisation of developing teeth was inhibited by denervation in Tiapia Mariae

fish, a polyphyodont species (Tuisku & Hildebrand 1994).

Other studies have suggested that the density o f nerves innervating the tooth primordia and the dental pulp are reminiscent o f an older, protective phylogenetic function such as nociception (Lumsden 1979, Northcutt & Cans 1983). The above studies suggest that animals of earlier phylogenetic origin have retained the potential to regenerate limb buds and fins (amphibian and fishes) and that the nervous system controls this regenerative process. Mammalians appear to have lost this ability or have down- regulated the expression to function in homeostatic maintenance and, perhaps, wound healing.

Wound healing -The morphologic responses of CGRP-ir nerve fibres to injury and healing have been extensively studied and include nerve sprouting and altered levels of CGRP in response to exposing the pulp (Khayat et al. 1988), cavity preparation (Kimberley & Byers 1988, Taylor et al. 1988), microabscess formation (Taylor & Byers 1990), and pulpotomy (Byers et al. 1990). Alongside these studies, evidence for important interactions between sensory nerve fibres and pulp cells is accumulating. For example SP, NKA, and CGRP have been shown to affect pulpal fibroblast proliferation

in vitro (Bongenhielm et al. 1995).

Studies o f healing in denervated teeth, or teeth replantated after extraction show that normal pulp healing and function after injury is dependent on sensory nerves (Kvinnsland et al. 1991, Byers & Taylor 1993, Nârhi & Byers 1999). Deneravtion also affects the differentation of cells into new odontoblasts, the function o f new or old odontoblasts and the rate of secondary dentin deposition in rat molars (Byers et al.

1992, Jacobson & Heyeraas 1996, Zhang & Fukuyama 1999).

voltage-gated, as well as nonvoltage-gated, agonist sensitive Ca channels (Lundgren and Linde, 1997). ATP can directly affect these channels, resulting in increased Ca uptake as a preparatory step in up-regulating odontogenesis.

There are also important interactions between sensory nerves and immunocompetent dendritic cells in the pulp (Bergenholtz et a l 1991). For example sensory nerves have a pronounced influence on the recruitment and transendothelial migration o f immunocompetent cells during experimental inflammation in teeth (Fristad et a l 1995, 1997).

Taken together these studies suggest that interactions between sensory nerves and pulp tissue are important for the survival o f pulp, the response o f pulp to injury, and the repair of pulp and dentin (Byers 1995, Zhang & Fukuyama 1999). In this context, it could be speculated that ATP and its receptors may also be involved in the tissue’s response to injury and repair. ATP acts as a growth factor for many cell types, including vascular smooth mucle cells (Wang et a l 1992, Abbracchio et a l 1995, Ehrling 1998). In other tissues, ATP released from activated fibroblasts as a result of mechanical stress might be important in fibroblast migration to wound areas (Grierson & Meldolesi (1995). Since I experiments of injujred pulp tissue nerves follow intact viable odonto- balsts (Byers & Nârhi 1999), ATP released from stretched odontoblasts may play a similar role in migration of fibroblasts or as a guide for nerve regeneration.

E) Cell-cell communication through wave propagation

Mechanical stimulation of many cells (such as airway epithelial cells, glial cells, endothelial cells, mast cells, liver cells and osteoblastic cell lines) have been shown to induce Ca^^ waves which propagate from the point of stimulation and travel across cell borders to adjacent cells (Osipchuk & Calahan 1992, Schlosser et a l 1996, Cotrina et al 1998, Jorgensen et a l 1997). In a recent study, Sauer et a l (2000) showed that

2+

mechanical strain applied to prostate cancer cells induced intracellular Ca wave spreading via ATP release, to a distance of approximately 300 pm.

Such mechanically induced signalling can occur through at least two pathways, one involves intracellular communication via gap junctions and the other is extracellular communication via release o f ATP and activation o f purinoceptors (Osipchuk and Cahalan 1992, Sauer et a l 2000).

Gap junctions have already been shown between odontoblasts (Ushiyama 1989). Odontoblasts may also use ATP release as a means of cell-to-cell signalling. Calcium waves may provide a means of transducing localised strain events into extended signalling cascades in distant cell layers that are not affected by the mechanical strain. If odontoblasts when stretched (either mechanically or by hypotonicity) releases ATP, this ATP can cause a Ca^^ wave propagation and activate other odontoblasts not directly stretched by the stimulus, thereby amplifying the original stimulus. We can hypothesize, therefore, that ATP may also play a role in intercellular signalling via a pathway independent of gap junctions.

Fig. 4.1 summarises the hypothetical roles for ATP in human dental pulp.

II. Future Studies

Anatomical studies

a) We found indirect evidence that potent ATPases are present on or around odontoblasts. The activity of these enzymes can be studied by measuring the inorganic (Pj) production using ATP as a substrate (Zingashin et a l 1995). Alternatively, these enzymes can be immunohistochemically localised. Antibodies are available against CD39, an ecto-apyrase shown to be expressed in a variety of tissues, and linked to sites o f purinergic signalling (Wang & Guidotti 1996 & 1998, Vlajkovic et a l 1998). The presence o f these enzymes on or around odontoblasts will provide further circumstantial evidence for the release of ATP from odontoblasts.

b) It has been shown that CGRP-ir nerve fibres in rat molars increase in number (nerve sprouting) in response to injury (Taylor et a l 1988). It would be interesting to investigate if the same reaction can be observed in PlXg-ir neurons.

ATP release

c) Odontoblast cell lines provide an easier experimental model as the cells are less fragile and can be grovm freely (McDougal et a l 1995). Mechanical or other stimulation o f odontoblast cell lines may provide useful information such as a) do these cells react to stimuli by releasing ATP, and b) whether the cell lines retain the same properties as adult odontoblasts in situ.

d) Stimuli that are present during inflammatory conditions, such as inflammatory mediators and/or algogenic substances (e.g. capsaicin), may also induce ATP release and could be tested using odontoblasts, either in situ or as cell lines.

e) Perfusion o f in vitro preparations of pulp tissue has been used to measure neuropeptide (CGRP) secretion from pulp afferents in response to stimuli which cause pain when injected into humans (Hargreaves et al. 1992 & 1995). This method can be adapted to investigate whether application o f stimuli to pulp tissue results in bulk release of ATP, e.g. from fibroblasts. It is known that direct mechanical stimulation of the exposed pulp activates C fibres in the pulp (Narhi 1985). Alternatively, ATP itself may cause release of substances such as inflammatory mediators from pulp. Preliminary experiments have already provided evidence that such release takes place (Cai et al. 2000) but more controlled studies are needed.

Functional studies

f) Functional studies are crucial in elucidating the exact role that ATP plays in dental pain pathways. One such study can be the application of ATP and other P2Xs agonists to dentin while recording nerve activity in normal and inflamed pulp. Another approach would be the application of other nociceptive stimuli to dentin while recording nerve activity in the presence o f P2Xg agonists or antagonists. For example, Sawynok & Ried (1997) reported that ATP and apm eATP augmented pain signals produced in the formalin test.

Spread o f Ca wave across

other cells

Fibroblast migration For wound healing

[Ca^^Ji for reparative dentinogenesis Nerve

/

Fig. 4.1: Schematic summary of the hypothetical effects of ATP released from odontoblasts.